全 文 ：马尾树科的系统位置:来自 rbcL基因
核苷酸序列的证据
陈 之 端 汪 小 全 孙 海 英 韩 英 张 志 宪 邹 喻 苹 路 安 民
(中国科学院植物研究所系统与进化植物学开放研究实验室 北京 100093)
SYSTEMATIC POSITION OF THE RHOIPTELEACEAE:
EVIDENCE FROM NUCLEOTIDE SEQUENCESOF rbcL GENE
CHEN Zhi-Duan WANG Xiao-Quan SUN Hai-Ying
HAN Ying ZHANG Zhi-Xian ZOU Yu-Ping LU An-Ming
(Laboratory of S ystematic and Evolut ionary Botany , Insti tu te of Botany , the Chinese Academy of Sciences , Beijing 100093)
Abstract Systematic posi tio n of the Rhoipteleaceae w as investigated using nucleotide se-
quences of the chloroplast gene rbcL.Two sets of parsimony analy ses w ere conducted , one
using Bauera as the outgroup and the o ther using Cercidiphyl lum-Liquidambar as out-
g roups.The results of these analyses are consistent.Rhoiptelea is allied wi th sampled Jug-
landaceous genera in the rbcL phylogeny , suggesting its close relationship with the Juglandaceae.
Key words Rhoipteleaceae;rbcL sequence;“higher” hamamelids
The Rhoipteleaceae , consisting of one species Rhoiptelea chi lantha Diels et Hand.-
Mazz., is sporadically distributed in southern China and northern Vietnam(Kuang , 1960).
It w as f irst described by Handel-Mazzet ti(1932)and w as placed in the U rt icales.However ,
few authors have followed Handel-Mazzet ti s treatment.Based on their studies of pollen
g rains and inf lorescences , Hjelmqvist(1948) and Erdtman(1952) considered that the
Rhoipteleaceae w as related to the Betulaceae.Many o ther authors believed that the Rhoipte-
leaceae had a close relationship with the Juglandaceae(e.g ., Oginuma et al., 1995;Zhang
et al., 1994;Thorne , 1992;Lu & Zhang , 1990;Chang , 1981;Cronquist , 1981;
Takhtajan , 1980;Manning , 1970).However , the cladistic analy sis based on pollen mor-
phology placed Rhoiptelea in an unresolved clade containing Nothofagus , Balanops , Betu-
laceae , Myricaceae , Casuarinaceae , and Juglandaceae(Zavada &Dilcher , 1986).
Sequences of the rbcL gene encoding large subunit of ribulose-1 , 5-bisphosphate car-
boxylase have been w idely utilized for resolving phylogenetic relationships at the family or
even at the genus level(Olmstead &Palmer , 1994;Chase et al., 1993).A couple of phylo-
genetic analyses have been conducted for the Hamamelidae using rbcL sequences(Gunter et
al., 1994;Chase et al., 1993;), but Rhoiptelea was not sampled therein.Therefore , the
objective of this study is to examine the systematic posi tio n of Rhoiptelea using DNA se-
quences of the rbcL gene.
1 Materials and methods
Twenty-f ive species were used for this study , representing 12 families of the Hamameli-
This research w as supported by National Natural Science Foundation of China(Grant No.39670056).
1997-09-22收稿 , 1997-11-21收修改稿。
植 物 分 类 学 报 36(1):1～ 7 (1998)
Acta Phytotaxonomica Sinica
dae.The rbcL gene w as sequenced for Rhoiptelea chil iantha(voucher specimens:Wang
Yin-Zheng 97188 and Lu Yuan-Xue 9709012 , deposited in PE Herbarium), while sequences
for the other species were withdraw n from the GenBank.The sampled taxa and their Gen-
Bank sequence accession numbers are listed in Table 1.
Table 1 rbcL sequences used in the present study
Family S pecies(abbreviations) Accession No. Reference cited
Betulaceae Aln us incana (ALNU) X56618 Bousquet et al., 1992
Betula papyr i fera(BETU) X56617 Bousquet et al., 1992
Carpinus caroliana(CARP) X56621 Bousquet et al., 1992
Corylus cornuta(CORY) X56619 Bousquet et al., 1992
Ostrya virginiana(OSTR) X56620 Bousquet et al., 1992
Cercidiphyllaceae Cercidiphyl lum japonica (CERC) L11673 Olmstead et al., 1992
Cunoniaceae Bauera rubioides(BAUE) L11174 Morgan &S olt is , 1993
Casuarinaceae Al locasuar ina ver ticil lata (ALLO) X69527 Maggia &Bousquet , 1994
Casuariana cunninghamia na(CNSU) X69528 Maggia &Bousquet , 1994
Gymnostoma webbia num (GYMN) X69531 Maggia &Bousquet , 1994
Fagaceae Castanea sativa(CAST) M94936 Frascaria et al., 1993
Chrysolepis sem pervirens(CHRY) U02727 Chase et al., 1993
Fagus americanus(FAGU) L13338 Chase et al., 1993
Nothofagus dombeya(NOTH) L13350 Chase et al., 1993
Quercus rubra(QUER) M58391 Bousquet et al., 1992
Hamamelidaceae Liqu idambar styraci f lua(LIQU) M58394 Bousquet et al., 1992
Juglandaceae Carya i l linoensi s(CARY) U00436 Gunter et al., 1994
J ug lans n igra(JUGL) U00437 Gunter et al., 1994
Pterocarya rhoi folia(PTER) U00439 Gunter et al., 1994
Moraceae Morus alba(MORU) L01934 Solti s et al., 1990
Myricaceae Comptonia peregrina(COMP) X69529 Maggia &Bousquet , 1994
Myrica ceri fera(MYRI) L01934 Maggia &Bousquet , 1994
Rhoipteleaceae Rhoiptelea chi liantha (RHOI) AF017687 the present paper
Ulmaceae U lm us alata(ULM U) U00441 Gunter et al., 1994
Urticaceae Pi lea pumi la(PILE) U00438 Gunter et al., 1994
Molecular techniques Total genomic DNA was ex tracted from fresh leaves of Rhoipte-
lea chiliantha , cultivated at the Institute of Botany , Chinese Academy of Sciences , Beijing ,
China , follow ing the protocol of Bousquet et al.(1990).
One forw ard direction amplification primer S2R (see Hoot et al., 1995)in the up-
st ream region of rbcL w as used.In the down st ream region of the gene rbcL , a reverse di-
rection amplif ication primer , rbcL-1494R w as designed.Other amplification and sequencing primers w ere designed
2 植 物 分 类 学 报 36 卷
based on conserved regions among published sequences for some species of hamamelids and gymnosperms.The primers w ith
thei r posit ions and base com positions are show n in Table 2.
Table 2 Location(position based on rbcL sequence of Betula papyri fera), base composition
of amplification and sequencing primers for the chloroplast gene rbcL
All primers were designed by Z.D.Chen
No. Name 5′sequence 3′ Locat ion
1 rbcL-1F ATG TCA CCA CAA ACA GAA ACT 1-21
2 rbcL-774F AGA AT T GGG AGT TCC TAT CGT 774-794
3 rbcL-196R ATG TAC CAG TAG AAG ATT CAG 196-176
4 rbcL-414R CAA ATC CTC CAG ACG TAG AGC 414-394
5 rbcL-638R CGC ATA AAT GGT TGG GAA TTC 638-618
6 rbcL-991R CGG TAC CAG CGT GAA TAT GAT 991-971
7 rbcL-1208R CCG AAT TGT AGT TAC GGA ATC 1208-1188
8 rbcL-1494R GAT TGG GCC GAG TT T AAT TAC 1494-1474
Polymerase Chain Reaction(PCR)amplification w as conducted in a thermocycler(Perkin
Elmer 9600).Primers S2R and rbcL-1494R were used to amplify a segment using the PCR.
The PCR products w ere purif ied by WizardR PCR preps DNA Purification Sy stem
(Promega), and then sequenced directly on LKB DNA sequencing station using SILVER
SEQUENCETM DNA Sequencing System(Promega)and a set of sequencing primers.
Phylogenetic analysis Sequences aligned using HIBIO DNASISTM(Hitachi)were im-
po rted into the PAUP prog ram(version 3.1.1 , Sw offo rd , 1993)for parsimonious analysis.
Two separate analyses w ere conducted , one using Bauera of Cunoniaceae as the outg roup ,
while the other using Cercidiphy llum and Liquidambar of “ lower” hamamelids as out-
g roups.For each analysis , 100 replications of RANDOM addition w ere conducted using
heuristic searches with TBR branch sw apping and MULPARS to save all equally most parsi-
monious t rees.Boo tst rap analyses of 500 replicat ions were performed to show relative sup-
po rts fo r individual clades.
2 Results
The rbcL gene in Rhoiptelea chiliantha is 1428 bp long.In the data matrix , 237 sites
w ere found to be variable , 159 of w hich were potentially informative , w ith 25 sites(15.7%)
at the first codon posi tion , 11 si tes(6.9%)at the second , and 123 sites(77.4%)at the
third.The absolute distance and mean distance betw een Rhoiptelea chi liantha and each of
o ther tax a were presented in Table 3.
Table 3 Absolute distances and mean distances of pairwise sequences betw een
Rhoiptelea chiliantha and each of o ther 24 species.Abbreviations as in Table 2
ALN U BET U CARP CAST CASU COM P CORY FAGU
Absolu te distance 30 35 43 45 50 38 36 51
Mean distance 0.021 0.025 0.030 0.032 0.035 0.027 0.025 0.036
GYMN OSTR PETE LIQU CERC CARY ULMU PILE
Absolu te distance 42 42 10 71 75 10 77 86
Mean distance 0.029 0.029 0.007 0.050 0.053 0.007 0.054 0.060
NOTF MORU CHRY BAUE MYRI JUGL QUER ALLO
Absolu te distance 54 62 47 75 37 10 49 49
Mean distance 0.038 0.043 0.033 0.053 0.026 0.007 0.034 0.034
1 期 陈之端等:马尾树科的系统位置:来自 rbcL 基因核苷酸序列的证据 3
Fig.1 One of the tw o shortest t rees found in parsimony analysis using Bauera as an outgroup(a),
and the strict consensus t ree of the tw o most parsimonious t rees(b).Numbers above branches
in(a)indicate branch lengths.Numbers below b ranches in(b)indicate bootst rap values.
Taxon abbreviat ions as in Table 2
The analysis employing Bauera as the outg roup resulted in tw o most parsimonious t rees
of 525 steps w ith a consistency index(CI)of 0.623.Fig.1b is the consensus t ree.Rhoipte-
lea is closely related to Peterocarya , Juglans and Carya of the Juglandaceae.They fo rm a
w ell supported clade w ith 9 base substitutions , a bootst rap value of 100%.The Fagaceae
sensu lato including the genus Nothofagus is not suppo rted by the rbcL phylogeny , but
Castanea , Quercus and Chrysolepis are united by 24 base substitutions , a boo tst rap value of
100%.Fagus is isolated f rom the above clade.The Nothofagaceae is the sister g roup of all
o ther “ higher” hamamelid families , and these families consist of an unresolved clade as
show n in the st rict consensus tree(Fig.1b).The monophyly of the Casuarinaceae and M yri-
caceae each is st rong ly suppo rted w ith a bootstrap value of 100%.The genera of the Betu-
laceae fo rm a clade , consist ing of two subclades.The first subclade including Cory lus ,
Carpinus and Ostrya is w ell supported , whereas the second subclade of Alnus and Betula is
not well supported , with only 3 base substi tutions and a boo tst rap value of 75%.
The analysis using Cercidiphyl lum and Liquidambar as outg roups generated two most
parsimonious t rees with the same tree topologies as the one using Bauera as outg roup.
4 植 物 分 类 学 报 36 卷
3 Discussion
The previous studies have suggested that Rhoiptelea is closely related to the Juglan-
daceae(Oginuma et al., 1995;Zhang et al., 1994;Tho rne , 1992;Lu &Zhang , 1990;
Chang , 1981;Takhtajan , 1980;Manning , 1970).In the rbcL phylogeny , the clade con-
taining Rhoiptelea and the sampled genera of the Jug landaceae is supported with a bootst rap
of 100%.Therefore , this study of fers strong support fo r the suggestion.
Morphologically , the important similarity between the Rhoipteleaceae and the Juglan-
daceae is the pinnate leaf.A diagnostic character is hermaphroditic f lowers with anatropous
and bitegmic ovules in the Rhoipteleaceae , but monoecious with ortho tropous and unitegmic
ovules in the Juglandaceae(Chang , 1981).However , hermaphroditic f lowers are also found
in Pterocarya of the Juglandaceae(Su &He , 1984).In addi tion , bisexual inf lo rescences are
common in the Juglandaceae(Lu &Zhang , 1990).Phy tochemically , Jiang and Zhou(1990)
reported that the composition and content of fat ty acid in the Rhoipteleaceae are very similar
to those in Engelhardia , Platycarya and Cyclocarya of the Juglandaceae , but dif ferent from
those in the Betulaceae , Fagaceae and Ulmaceae.Recent karyomorphological study of
Rhoiptelea show ed that its basic chromosome number is x=16 as that in the Juglandaceae
(Oginuma et al., 1995).Therefore , the above evidence also supports the close relat ion-
ships of Rhoiptelea w ith the juglandaceous genera , cong ruent with the result of this study.
The so-called “higher” hamamelids , including Betulaceae , Ticodendraceae , Fagaceae
sensu lato , Juglandaceae , Myricaceae and Casuarinaceae , are considered more closely related
to Rosidae than to “ low er” Hamamelidae , and the Cunoniaceae is considered to be a sister
g roup of the “higher” hamamelids(Chase et al., 1993;Hufford , 1992;Zavada &Dilcher ,
1986).Therefore , we also used Bauera of the Cunoniaceae as the outg roup in the present
study , in addition to employing Cercidiphyl lum and Liquidambar of the “ lower”
hamamelids as the outgroups.These tw o analyses resul ted in the same arrangements about
the “higher” hamamelid groups.
Although w e increased the taxon samples , the family relationships in this analysis are
very similar to those suggested from the previous studies by Chase et al.(1993)and Gunter
et al.(1994)based on the sequence data f rom rbcL gene , and Manos et al.(1993)based
on the analysis of the rest riction site variation in the inverted region of the chloroplast DNA.
Acknowledgement We thank D r.Li Jian-hua at Univ ersity o f New Hampshire , USA for helpful comments
and improving the Eng lish of the manuscript , and our co lleagues Zhang Zhi-yun and Chen Ji-w ang for provid-
ing the fresh leaves of Rhoiptelea chiliantha.
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摘要 本文根据叶绿体基因 rbcL的核苷酸序列证据讨论了单型科———马尾树科的系统关系。数据矩
阵中包括了马尾树科及其它“高等”金缕梅类各科 ,并分别利用 Cunoniaceae科的 Bauera 属 , “低等”金缕
梅类植物连香树属和枫香树属作为外类群进行分支分析。两种分析的结果是一致的。在 rbcL 基因树
上 ,马尾树属和被用于分析的胡桃科各属紧密地结合在一起 ,支持马尾树科和胡桃科有非常近的亲缘关
系。
关键词 马尾树科;rbcL序列;“高等”金缕梅类
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1 期 陈之端等:马尾树科的系统位置:来自 rbcL 基因核苷酸序列的证据 7